| 研究課題/領域番号 |
25870525
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| 研究種目 |
若手研究(B)
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| 研究機関 | 長崎大学 |
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研究代表者 |
カレトン リチャード 長崎大学, 熱帯医学研究所, 准教授 (10503782)
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| 研究期間 (年度) |
2013-04-01 – 2015-03-31
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| キーワード | Malaria / Genomics / Genetics / Bioinformatics / LGS / WGS / Gametocyte / Vaccine |
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| 研究概要 |
To discover novel transmission blocking vaccine proteins in malaria parasites using a combination of classical genetic linkage analysis (LGS) and whole genome quantitative resequencing. We planned on using only the rodent malaria parasite system, but have now also embarked on studies with Plasmodium falciparum. We have concentrated on the following three aspects:
i) Initiating strain-specific transmission-blocking immunity in Plasmodium yoelii. We have performed numerous experiments using natural immunity to block transmission in P. yoelii. We have shown that there is a sharp decrease in the infectivity of gametocytes throughout the course of an infection in mice, and, using IFN-Gamma and MyD88 KO mice, that this is independent of the innate immune response. We have used sera transfer to demonstrate that sera from mice with long-term infections supresses the ability of early-infection stage gametocytes to transmit, and that this suppression has a strain-specific component.
ii) We are currently investigating the feasibility of performing LGS experiments with P. falciparum. Using the strains 3D7 and HB3, we have cultured pure gametocytes, and purified crude protein, to produce immune sera in rabbits. We have acquired the uncloned recombinant progeny of a cross between these two strains.
iii) We have developed bioinformatics and statistic techniques capable of identifying statistically significant deviations in the relative frequency of SNP markers measured in whole genome resequencing analyses of recombinant progeny populations of malaria parasites following selection.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
1: 当初の計画以上に進展している
理由
We initially intended to identify strains of the rodent malaria parasite P. yoelii that can induce transmission blocking immunity specific to the strains. We have achieved this through the demonstration that late-infection stage (from mice infected two weeks previously) sera from P. yoelii strain CU, can, when transferred to mice with early infections (Day 3 post-infection) significantly reduce the transmission of parasites to mosquitoes (measured as the percentage of infected mosquitoes, and the number of oocysts per infected mosquito), compared to naive sera. Sera transfer from mice infected with late-stage infections of another strain, P. yoelii 17X1.1pp, did not significantly reduce transmission of early stage infections of P. yoelii CU, but did supress infections of the homologous strain. We have produced a genetic cross between these two strains for LGS analysis.
We have developed a robust bioinformatics and statistical analysis protocol for the interrogation of selected progeny. We have tested this methodology on crosses selected for growth rate differences and immunity to the blood stages.
Finally, we have begun investigations into strain specific immunity using the human malaria parasite P. falciparum. We plan to perform a full LGS with this parasite using in vitro culture and mosquito transmission. We have successfully purified gametocytes of 3D7 and HB3 strains, and prepared crude antigen for production of transmission blocking sera. We have also acquired the uncloned recombinant progeny of a cross between the two parasite strains for downstream LGS experiments.
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| 今後の研究の推進方策 |
During the next year, we plan to perform LGS analyses on transmission-blocking immunity selected cross progeny for both P. yoelii (in mice) and P. falciparum (in vitro culture).
i) Plasmodium yoelii. The first priority is to optimise the strain specific immunity blocking protocol. Currently, our experiments involve the transfer of sera from mice with late stage infections of parasites to mice with early stage infections. This results in a significant reduction in both the percentage of mosquito infected and the numbers of oocysts per infected mosquito, but does not completely block transmission. For the LGS experiments, it is essential that transmission be blocked as thoroughly as possible. We will, therefore, attempt to maximise the transmission-blocking efficacy of immune sera by a) immunising mice directly with crude antigen derived from purified gametocytes of each strain, and b) switching to membrane feeding rather than in vivo feeding of mosquitoes. Following this optimisation, we will perform the LGS experiments, involving selection of recombinant progeny, whole genome resequencing, and vaccine candidate identification.
ii) Plasmodium falciparum. We have purified gametocytes from 3D7 and HB3 strains, and have produced protein extracts from them. We now plan to immunise rabbits with these proteins to produce immune sera which we will test for it’s ability to block transmission to mosquitoes in a strain specific manner. Following this, we will attempt to perform LGS analyses using these immune sera and the uncloned recombinant progeny of a cross between the two strains
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| 次年度の研究費の使用計画 |
One experiment was postponed due to time constraints.
This experiment was to be conducted in February 2014 and was to cost 260,000 yen. We will now carry out this experiment in May 2014.
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